Despite the great advances made in our understanding of synapse development at the neuromuscular junction, the roles of several key proteins remain unclear. The proposed experiments will use the unique advantages offered by zebrafish genetics and development to identify the roles of rapsyn, acetylcholine receptor, MuSK and beta-dystroglycan in synapse formation in vivo. Zebrafish offers tremendous advantages over mammalian in vitro and in vivo systems. Studies using in vitro mammalian expression systems and cultured myotubes have both been hampered by the inability to study bona-fide synapses. The in vivo studies have been limited by the inability of the mouse knock-outs to survive through the period of synapse formation. In the case of receptor knock-out, for example, the consequences are so severe that no studies have been able to address the consequences of receptor-less development. By contrast, functional knock-outs of acetylcholine receptor, rapsyn, and MuSK have been identified in mutant lines of zebrafish. These fish were originally identified on the basis of swimming abnormalities that reflect direct consequences of knock-outs of each of these key synaptic proteins. This analysis is possible in zebrafish because, unlike their mammalian counterparts, these mutant animals die well after synapse formation is completed and the animal behavior can be assessed. To date our findings have revealed most unexpected roles for the acetylcholine receptor and for rapsyn in governing synapse development and function. In particular, we have found that the receptor likely plays a key role in localizing rapsyn to the synapse and rapsyn plays a critical role in regulating receptor function. Additionally, our studies have provided new predictions for human neuromuscular diseases, one of which has been confirmed on patients afflicted with rare forms of myasthenia gravis. We are confident that this model system will, through its many unique advantages, resolve some of the outstanding paradoxes involving the roles of signaling molecules in synapse formation.